1. Field of the Invention
The present invention relates generally to the fields of cell surface receptors and their functions. More particularly, it concerns the generation of engineered receptor molecules that are constitutively active in the absence of the cytokine, hormone or molecule that normally activates the receptor. Disclosed are various DNA, protein and cellular compositions and methods of making and using constitutively active receptors, particularly, receptors engineered to form multimers.
2. Description of the Related Art
Cytokines are a group of peptide hormones that interact with cell surface receptors to signal specific biological effects. The tyrosine kinase receptors have a consensus tyrosine kinase sequence in the cytoplasmic domain that is involved in the signal transduction mechanism. Other types of receptors signal by mechanisms that are less well understood, although there is evidence for tyrosine phosphorylation in some receptors.
The erythropoietin receptor (EPOR) recognizes a glycoprotein hormone ligand. This hormone (erythropoietin) is required for the survival, proliferation and differentiation of committed erythroid progenitors. Other members of the cytokine family to which this receptor belongs include the receptors for hematopoietic growth factors such as interleukins, colony-stimulating factors and growth hormones. Unlike the tyrosine kinase receptors, the mode of signalling activity of these cytokine receptors is not known. However, it has been suggested that dimerization or oligomerization of these receptors plays an important role in the signal mechanism (Oyashi et al., 1994).
Previous studies have also shown that a mutation in the EPOR which converts an arginine to cysteine at position 129 confers constitutive expression and induces stable receptor homooligomers (Oyashi et al., 1994; Longmore and Lodish, 1991). Chimeric receptors have also been expressed by the same group in which the cytoplasmic and extracellular domains of the EPOR and the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, have been fused to form chimeric receptors that respond to the extracellular domain ligand by the response mechanism of the cytoplasmic domain.
Tumor necrosis factor (TNF) is a cytokine mediator which initially showed much promise as an antineoplastic agent, since the protein specifically destroys transformed cells in vitro, and causes the hemorrhagic necrosis of transplantable tumors in vivo without killing normal cells. Subsequently, however, it was demonstrated that TNF has many toxic effects in living animals. Specifically, it appears to be a central mediator of endotoxic shock. Unfortunately, this has limited the therapeutic application of TNF. No human tumor has ever been successfully treated with the protein.
All of the biological effects of TNF are mediated by two types of cell membrane receptor. The larger of these, a 75 kD glycoprotein, transduces the proliferative effect of TNF, although some cytotoxic activity may be generated through this molecule as well. The smaller receptor, a 55 kD cell surface glycoprotein, bears some homology to the larger TNF receptor in the region of the extracellular domain, but has an entirely different cytoplasmic domain. It therefore generates a different signal when activated by ligand binding. It is the 55 kD cell surface receptor that appears to be chiefly responsible for the induction of TNF-mediated cytotoxicity.
TNF is a trimeric molecule. The TNF receptors exist as a mixed population of monomers on the surface of virtually all somatic cells. TNF initiates signals through both of the two types of receptor by cross-linking three identical monomeric subunits (either three 75 kD subunits or three 55 kD subunits) on the cell surface. The juxtaposition of the monomers leads to generation of a signal through a process that is not completely understood. It is clear, however, that the cross-linking of monomers, and not the engagement of TNF per se, is the important event in signal transduction. Therefore, anti-receptor antibodies can substitute for TNF, generating an agonist signal. There still exists a need however, for a mechanism of inducing the cytotoxic TNF response without inducing the side effects that are caused by administration of TNF.